This invention relates to a process for measuring an unsaturated iron-binding capacity in serum. More particularly, this invention relates to an improved process for measuring an unsaturated iron-binding capacity in serum rapidly and easily by using a special buffering agent which has weak coordinate bonding strength particularly with iron while having suitable bonding properties with iron in the case of using a chelate color-forming reagent.
Iron exists in a living body as hemoglobin in a red blood corpuscle in an amount of about 2/3 of iron and as stored iron in an amount of the rest 1/3 of iron in the liver, the spleen, the bone-marrow, and the like. The total weight of iron in a human body is about 4 g. The iron is lost in an amount of about 1 mg per day by peeling of mucous membrane from the intestine, removal of the skin, and the like and is hardly lost by excretion of urine. Therefore, the balance can be maintained by absorbing about 1 mg of iron from food every day. All the iron in serum is physiologically bound to transferrin which is a kind of globulin and is transferred. Transferrin is a kind of protein having a molecular weight of about 90,000 and has a capacity to bind two iron atoms per mole in the ferric form. Transferrin is generally present in serum in the form of the so-called serum iron wherein transferrin binds iron in an amount of 1/3 thereof and the rest 2/3 is present as transferrin not binding iron. The amount of transferrin not binding iron is measured as the unsaturated iron-binding capacity in serum.
Such iron in serum and the unsaturated iron-binding capacity relate to the metabolism of hemoglobin and the measurement of such items is indispensable for a differential diagnosis of anemic persons.
There have practically been used various methods for measuring the unsaturated iron-binding capacity which has a clinically important meaning. For example, there is a method wherein a minute amount of iron is gradually added for direct photoelectric colorimetry by using a principle that iron-transferrin produces a pink color [the Rath and Finch method: J. Clin. Invest, 28, 79-85 (1949)]. But this method is disadvantageous in that it is necessary to use a large amount of sample serum. There is also a method comprising adding an excess iron to serum, adsorbing iron not bound to transferrin by adding a magnesium carbonate powder thereto, separating the magnesium carbonate by a centrifugal separating procedure, and measuring the iron in a supernatant liquid [the Ramsay method: Clin. Chim. Acta, 2, 221-226 (1957)]. But this method is complicated and disadvantageous. There is also a method wherein radioactive Fe.sup.59 is used, but since a non-sealed isotope is used, the use of it is very limited.
On the other hand, there is a method comprising adding an alkaline buffer solution to serum, adding iron to the mixture to bind transferrin to iron, and colorimetrically determining the residual iron to measure the unsaturated iron-binding capacity [the Schade method: Proc. Soc. Exp. Biol. & Med., 87, 443-448 (1954)]. More in detail, the Schade method comprises adding 1.0 ml of serum to 2.0 ml of a 1.0M tris buffer solution (pH=8.1), allowing the resulting mixture to stand for 5 minutes, adding 1.0 ml of an ammonium ferrous sulfate solution (3.51 mg/dl) containing 0.5% by weight of ascorbic acid to the resulting mixture, measuring absorbance at 535 nm by using a photoelectric colorimeter (blank), adding a drop of 0.5% bathophenanthroline sulfonic acid-Na.sub.2 salt for color formation, allowing to stand at 25.degree. C. for 10 minutes or more, and measuring absorbance at 535 nm. It is said that serum iron releases iron from transferrin at pH 4.0 or lower while the bonding between iron and transferrin is strong and stable at the alkaline side. But when a chelate color-forming reagent such as bathophenanthroline is added, dissociation of transferrin and iron begins to take place at about pH 7.0 or lower. But when the pH is 7.5 or higher, no dissociation of transferrin and iron takes place even if bathophenanthroline is present. Therefore, according to the Schade method, it is essential to conduct the measurement at the pH of 7.5 or higher. On the other hand, the color-forming reaction rate is maximum at about pH 5.0 in the case of using bathophenanthroline as a color-forming reagent. The color-forming rate is slowed down when the pH is either higher or lower than that pH (about 5.0). When bathophenanthroline is used as a color-forming reagent in the Schade method, it is necessary to make the pH of the solution about 7.5 to 8.5 considering the stability of serum iron. But when pH=8.1 is selected in practice, it takes about 10 minutes for the color formation. Thus, the shortening of the color-formation in the Schade method is demanded strongly in order to use an auto analyzer, although the Schade method is a very simple method without using centrifugal separation, removal of proteins and complicated procedures.